Isolation of an oxygen-evolving Photosystem II preparation containing only one tightly bound calcium atom from a chlorophyll b-deficient mutant of rice

Oxygen-evolving Photosystem II (PS II) particles were prepared from the thylakoid membranes of a chlorophyll b-less rice mutant, which totally lacks light-harvesting chlorophyll a/b proteins, after solubilization with β-octylglucoside. The preparation was essentially free of Photosystem I as judged from its low-temperature fluorescence spectrum and polypeptide composition. The PS II particles contained all the major subunit polypeptides of the PS II reaction center core complexes and the three extrinsic proteins related to oxygen evolution. The relative abundances of the 33, 21 and 15 kDa proteins were 100, 64 and 20%, respectively, of the corresponding proteins in the mutant thylakoids. The chlorophyll-to-Q_A ratio was 53 and there was only one bound Ca²⁺ per Q_A. Thus, one of the two bound Ca²⁺ present in the oxygen-evolving PS II membrane preparations from wild-type rice (Shen J.-R., Satoh, K. and Katoh, S. (1988) Biochim. Biophys. Acta 933, 358–364) is missing. The mutant PS II particles were highly active in oxygen evolution in the absence of exogenously added Ca²⁺, although addition of 5 mM Ca²⁺ enhanced the activity by 30%. When the 21 and 15 kDa proteins were supplemented to the particles, the Ca²⁺-effect disappeared and the rate of oxygen evolution increased to a level exceeding 1000 μmol O₂ per mg chlorophyll per h. The results indicate that the number of Ca²⁺ needed to promote a high rate of oxygen evolution is one per PS II in higher plants.     

Inactivation and Calcium-Dependent Reactivation of Oxygen Evolution in Photosystem II Preparations Treated at pH 3.0 or with High Concentrations of NaCl

A brief treatment at pH 3.0 of Photosystem II (PS II) membranescontaining two bound Ca²⁺ from rice resulted in strong suppressionof oxygen evolution concomitant with extraction of one Ca²⁺and the lost activity was restored on addition of 50 mM Ca²⁺.However, inactivation of oxygen evolution by low pH-treatmentof oxygen-evolving PS II complexes containing only one Ca²⁺from a rice chlorophyll b-deficient mutant was not associatedwith extraction of the bound Ca²⁺, although oxygen evolutionwas markedly enhanced by the addition of Ca²⁺ to the treatedcomplexes. Thus, the acid-inactivation of oxygen evolution cannotbe related to extraction of Ca²⁺. On the other hand, low pH-treatmentwas found to share the following common features with NaCl-treatmentwhich also causes a Ca²⁺-reversible inactivation of oxygen evolution.(1) Exposure of PS II membranes to pH 3.0 resulted in solubilizationof the 23 and 17 kDa extrinsic proteins, although the releasedproteins rebound to the membranes when pH was raised to 6.5.(2) There was an apparent heterogeneity in the binding affinityof Ca²⁺ effective in restoration of the oxygen-evolving activity.(3) Low pH-treated preparations required a higher concentrationof Ca²⁺ for the maximum reactivation of oxygen evolution thandid NaCl-washed preparations. This was also the case with Sr²⁺,which stimulated oxygen evolution of both low pH-treated andNaCl-washed PS II membranes to smaller extents. When the extrinsic23 and 17 kDa proteins had been removed, however, Ca²⁺ concentrationdependence of oxygen evolution in low pH-treated membranes becamesimilar to that in NaCl-washed PS II preparations and the changeswere largely reversed by rebinding of the two proteins. Theseresults strongly suggest that low pH-treatment and NaCl-washinvolve similar mechanisms of Ca²⁺-dependent reactivation. ¹ Present address: Solar Energy Research Group, The Instituteof Physical and Chemical Research (RIKEN), Wako, Saitama, 351-01Japan (Received August 27, 1990; Accepted February 12, 1991)     

Molecular mechanism of high-temperature-induced inhibition of acceptor side of Photosystem II

High-temperature-induced inhibition of the acceptor side of Photosystem II (PS II) was studied in tobacco thylakoids using oxygen evolution, chlorophyll a (Chl a) fluorescence and redox potential measurements. When thylakoids were heated at 2 °C/min from 25 to 50 °C, the oxygen evolving complex became inhibited between 32 and 45 °C, whereas the acceptor side of PS II tolerated higher temperatures. Variable Chl a fluorescence decreased more slowly than oxygen evolution, suggesting that transitions between some S-states occurred even after heat-induced inhibition of the oxygen evolving activity. 77 K emission spectroscopy reveals that heating does not cause detachment of the light-harvesting complex II from PS II, and thus the heat-induced increase in the initial F₀ fluorescence is due to loss of exciton trapping in the heated PS II centers. Redox titrations showed a heat-induced increase in the midpoint potential of the Q_A/Q_A ^-) couple from the control value of –80 mV to +40 mV at 50 °C, indicating a loss of the reducing power of Q_A ^-). When its driving force thus decreased, electron transfer from Q_A ^-) to Q_B in the PS II centers that still could reduce Q_A became gradually inhibited, as shown by measurements of the decay of Chl a fluorescence yield after a single turnover flash. Interestingly, the heat-induced loss of variable fluorescence and inhibition of electron transfer from Q_A ^-) to Q_B could be partially prevented by the presence of 5 mM bicarbonate during heating, suggesting that high temperatures cause release of the bicarbonate bound to PS II. We speculate that both the upshift in the redox potential of the Q_A/Q_A ^-) couple and the release of bicarbonate may be caused by a heat-induced structural change in the transmembrane D1 or D2 proteins. This structural change may, in turn, be caused by the inhibition of the oxygen evolving complex during heating.     

PsbU provides a stable architecture for the oxygen-evolving system in cyanobacterial photosystem II

PsbU is a lumenal peripheral protein in the photosystem II (PS II) complex of cyanobacteria and red algae. It is thought that PsbU is replaced functionally by PsbP or PsbQ in plant chloroplasts. After the discovery of PsbP and PsbQ homologues in cyanobacterial PS II [Thornton et al. (2004) Plant Cell 16, 2164-2175], we investigated the function of PsbU using a psbU deletion mutant (DeltaPsbU) of Synechocystis 6803. In contrast to the wild type, DeltaPsbU did not grow when both Ca2+ and Cl- were eliminated from the growth medium. When only Ca2+ was eliminated, DeltaPsbU grew well, whereas when Cl- was eliminated, the growth rate was highly suppressed. Although DeltaPsbU grew normally in the presence of both ions under moderate light, PS II-related disorders were observed as follows. (1) The mutant cells were highly susceptible to photoinhibition. (2) Both the efficiency of light utilization under low irradiance and the chlorophyll-specific maximum rate of oxygen evolution in DeltaPsbU cells were 60% lower than those of the wild type. (3) The decay of the S2 state in DeltaPsbU cells was decelerated. (4) In isolated PS II complexes from DeltaPsbU cells, the amounts of the other three lumenal extrinsic proteins and the electron donation rate were drastically decreased, indicating that the water oxidation system became significantly labile without PsbU. Furthermore, oxygen-evolving activity in DeltaPsbU thylakoid membranes was highly suppressed in the absence of Cl-, and 60% of the activity was restored by NO3- but not by SO4(2-), indicating that PsbU had functions other than stabilizing Cl-. On the basis of these results, we conclude that PsbU is crucial for the stable architecture of the water-splitting system to optimize the efficiency of the oxygen evolution process.     

Probing the functional role of Ca2+ in the oxygen-evolving complex of photosystem II by metal ion inhibition

Photosynthetic oxygen evolution in photosystem II (PSII) takes place in the oxygen-evolving complex (OEC) that is comprised of a tetranuclear manganese cluster (Mn4), a redox-active tyrosine residue (YZ), and Ca2+ and Cl- cofactors. The OEC is successively oxidized by the absorption of 4 quanta of light that results in the oxidation of water and the release of O2. Ca2+ is an essential cofactor in the water-oxidation reaction, as its depletion causes the loss of the oxygen-evolution activity in PSII. In recent X-ray crystal structures, Ca2+ has been revealed to be associated with the Mn4 cluster of PSII. Although several mechanisms have been proposed for the water-oxidation reaction of PSII, the role of Ca2+ in oxygen evolution remains unclear. In this study, we probe the role of Ca2+ in oxygen evolution by monitoring the S1 to S2 state transition in PSII membranes and PSII core complexes upon inhibition of oxygen evolution by Dy3+, Cu2+, and Cd2+ ions. By using a cation-exchange procedure in which Ca2+ is not removed prior to addition of the studied cations, we achieve a high degree of reversible inhibition of PSII membranes and PSII core complexes by Dy3+, Cu2+, and Cd2+ ions. EPR spectroscopy is used to quantitate the number of bound Dy3+ and Cu2+ ions per PSII center and to determine the proximity of Dy3+ to other paramagnetic centers in PSII. We observe, for the first time, the S2 state multiline electron paramagnetic resonance (EPR) signal in Dy3+- and Cd2+-inhibited PSII and conclude that the Ca2+ cofactor is not specifically required for the S1 to S2 state transition of PSII. This observation provides direct support for the proposal that Ca2+ plays a structural role in the early S-state transitions, which can be fulfilled by other cations of similar ionic radius, and that the functional role of Ca2+ to activate water in the O-O bond-forming reaction that occurs in the final step of the S state cycle can only be fulfilled by Ca2+ and Sr2+, which have similar Lewis acidities.     

The inhibitory mechanism of Cu(II) on the Photosystem II electron transport from higher plants

In a previous paper, we reported that Cu(II) inhibited the photosynthetic electron transfer at the level of the pheophytin-Q_A-Fe domain of the Photosystem II reaction center. In this paper we characterize the underlying mechanism of Cu(II) inhibition. Cu(II)-inhibition effect was more sensitive with high pH values. Double-reciprocal plot of the inhibition of oxygen evolution by Cu(II) is shown and its corresponding inhibition constant, K_i, was calculated. Inhibition by Cu(II) was non-competitive with respect to 2,6-dichlorobenzoquinone and 3-(3,4-dichlorophenyl)-1,1-dimethylurea and competitive with respect to protons. The non-competitive inhibition indicates that the Cu(II)-binding site is different from that of the 2,6-dichlorobenzoquinone electron acceptor and 3-(3,4-dichlorophenyl)-1,1-dimethylurea sites, the Q_B niche. On the other hand, the competitive inhibition with respect to protons may indicate that Cu(II) interacts with an essential amino acid group(s) that can be protonated or deprotonated in the inhibitory-binding site.Abbreviations BSA bovine seroalbumin - Chl chlorophyll - DCBQ 2,6-dichlorobenzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - MES 2-(N-morpholino)-ethanesulphonic acid - Pheo pheophytin - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - PS Photosystem - RC reaction center - Tricine N-[Tris(hydroxymethyl)-methyl]-glycine     

The time course of photoinactivation of photosystem II in leaves revisited

Since photosystem II (PS II) performs the demanding function of water oxidation using light energy, it is susceptible to photoinactivation during photosynthesis. The time course of photoinactivation of PS II yields useful information about the process. Depending on how PS II function is assayed, however, the time course seems to differ. Here, we revisit this problem by using two additional assays: (1) the quantum yield of oxygen evolution in limiting, continuous light and (2) the flash-induced cumulative delivery of PS II electrons to the oxidized primary donor (P700(+)) in PS I measured as a 'P700 kinetics area'. The P700 kinetics area is based on the fact that the two photosystems function in series: when P700 is completely photo-oxidized by a flash added to continuous far-red light, electrons delivered from PS II to PS I by the flash tend to re-reduce P700(+) transiently to an extent depending on the PS II functionality, while the far-red light photo-oxidizes P700 back to the steady-state concentration. The quantum yield of oxygen evolution in limiting, continuous light indeed decreased in a way that deviated from a single-negative exponential. However, measurement of the quantum yield of oxygen in limiting light may be complicated by changes in mitochondrial respiration between darkness and limiting light. Similarly, an assay based on chlorophyll fluorescence may be complicated by the varying depth in leaf tissue from which the signal is detected after progressive photoinactivation of PS II. On the other hand, the P700 kinetics area appears to be a reasonable assay, which is a measure of functional PS II in the whole leaf tissue and independent of changes in mitochondrial respiration. The P700 kinetics area decreased in a single-negative exponential fashion during progressive photoinactivation of PS II in a number of plant species, at least at functional PS II contents ≥6?% of the initial value, in agreement with the conclusion of Sarvikas et al. (Photosynth Res 103:7-17, 2010). That is, the single-negative-exponential time course does not provide evidence for photoprotection of functional PS II complexes by photoinactivated, connected neighbours.     

Structure and function of the PsbP protein of Photosystem II from higher plants

PsbP is a membrane extrinsic subunit of Photosystem II (PS II), which is involved in retaining Ca²⁺ and Cl⁻, two inorganic cofactors for the water-splitting reaction. In this study, we re-investigated the role of N-terminal region of PsbP on the basis of its three-dimensional structure. In previous paper [Ifuku and Sato (2002) Plant Cell Physiol 43: 1244–1249], a truncated PsbP lacking 19 N-terminal residues (Δ19) was found to bind to NaCl-washed PS II lacking PsbP and PsbQ without activation of oxygen evolution at all. Three-dimensional (3D) structure of PsbP suggests that deletion of 19 N-terminal residues would destabilize its protein structure, as indicated by the high sensitivity of Δ19 to trypsin digestion. Thus, a truncated PsbP lacking 15 N-terminal residues (Δ15), which retained core PsbP structure, was produced. Whereas Δ15 was resistant to trypsin digestion and bound to NaCl-washed PS II membranes, it did not show the activation of oxygen evolution. This result indicated that the interaction of 15-residue N-terminal flexible region of PsbP with PS II was important for Ca²⁺ and Cl⁻ retention in PS II, although the 15 N-terminal residues were not essential for the binding of PsbP to PS II. The possible N-terminal residues of PsbP that would be involved in this interaction are discussed.     

Importance of the N-terminal sequence of the extrinsic 23 kDa polypeptide in Photosystem II in ion retention in oxygen evolution

The function of the extrinsic 23 kDa polypeptide (OEC23) in Photosystem II (PS II) is to retain Ca(2+) and Cl(-) during the S-state turnover of the Mn cluster in photosynthetic oxygen evolution. Recombinant OEC23s from several plant species were produced in Escherichia coli to characterize the molecular mechanism of the OEC23 function then used in reconstitution experiments. One tobacco isoform, OEC23 (2AF), had much less oxygen-evolving activity than the spinach and cucumber OEC23s when PS II activities were reconstituted in salt-washed spinach PS II particles. The fact that the OEC23s had similar binding affinities for PS II particles suggests different ion-retention capacities for the individual OEC23s: The chimeric OEC23s produced between spinach and 2AF and those produced between cucumber and 2AF show that 58 N-terminal amino acid residues are important for PS II activity. Further dissection of the sequence and site-directed mutagenesis indicated the importance of 20 N-terminal amino acid residues for activity, in particular the asparagine at the 15th position. In spinach the N15D mutation decreased PS II activity, whereas in 2AF the D15N mutation increased it. This shows the importance of the N-terminal sequence of OEC23 in ion retention during the water-splitting process.     

Recognition of interaction between the donor electron-transfer chains of Photosystem II under conditions of partial inhibition of oxygen evolution

The electron-transfer pathway on the donor side of Photosystem (PS) II has been examined using unfractionated and inside-out thylakoid membrane vesicles. A number of treatments are identified which result in the inhibition of light-dependent oxygen evolution. The differential capacities of the exogenous donors diphenylcarbazide and NH₂OH to restore the PS II-mediated reduction of 2,6-dichlorophenolindophenol (DCIP) in the inhibited membranes is discussed in terms of multiple donor sites for the electron-transfer pathway on the oxidising side of PS II. We also present data which indicate that the donor chains are not isolated from each other but that an individual PS II reaction centre may be able to interact with several oxygen-evolving complexes. The implication of such an interaction to the mechanism of oxygen evolution is discussed.     

Inhibition of the Chloroplast Photochemical Reactions by Treatment of Wheat Seedlings with Low Concentrations of Cadmium: Analysis of Electron Transport Activities and Changes in Fluorescence Yield

The effect of treatment of wheat plants with Cd²⁺ ions on thephotochemical activity of the primary leaves was examined. Threeday-old etiolated seedlings were treated with Cd²⁺ ions for24 h in dark, and after this treatment the plants were grownin the light until the primary leaves were fully developed.Cd²⁺ ions (30–120 µM) induced a significant decreasein activities of both photosystem II and photosystem I. Theextent of the decrease in PS II activity was much greater thanthat in the PS I activity. Analysis of changes in the fluorescenceyield of chlorophyll also indicated that Cd²⁺ ions drasticallyaffect the photochemistry of photosystem II. Cd²⁺ ions induceddecrease in the rates of photoreduction of 2,6-dichlorophenolindophenol even in the presence of the exogenous electron donor,hydroxylamine, both in Tris-treated and untreated chloroplasts.This result suggests that the site of inhibition is near thesite of donation of electrons by hydroxylamine. Treatment withCd²⁺ ions impairs the electron transport system on the reducingside of PS II. The decrease in the fluorescence yield of Chi is less than that in the evolution of O₂ mediated by oxidizedphenylenediamine. This difference may be a result of inhibitionon the reducing side of PS II. In addition to inhibition onthe reducing side, Cd²⁺ ions may affect the oxidizing side ofPS II. A comparative study of the rates of evolution of O₂ withp-benzoquinone and dichloro-p-benzoquinone as electron acceptorswas performed since the halogenated benzoquinones have beenshown to accept electrons from both active and inactive centersof photosystem II while some of the benzoquinones accept electronsonly from active centers. The results suggest that Cd²⁺ ionsinduced a marginal increase in the number of inactive reactioncenters in PS II. Analysis of light-saturation-kinetics of theevolution of O₂ catalysed by PS II indicates a reduction inthe size of the antennae as well as in the concentration ofthe active (-type) reaction centers of PS II. Thus, the Cd²⁺-inducedeffects on the photochemistry of PS II involve changes on thereducing side of PS II as well as possible changes in the sizesof the populations of active and inactive centers. Thus, short-termexposure to Cd²⁺ ions during establishment of seedlings hasa severely detrimental effect on photochemical activities inchloroplasts. (Received October 17, 1990; Accepted July 3, 1991)     

The 16 and 23 kDa extrinsic polypeptides and the associated Ca2+ and Cl- modify atrazine interaction with the photosystem II core complex

The oxygen evolving complex of photosystem II (PS II) contains three extrinsic polypeptides of approximate molecular weights 16, 23 and 33 kDa. These polypeptides are associated with the roles of Cl^-, Ca²⁺ and Mn²⁺ in oxygen evolution. We have shown that selective removal of 16 and 23 kDa polypeptides from the above complex by NaCl washing of PS II enriched membrane fragments renders the PS II core complex more susceptible to the herbicide atrazine. On the other hand, when both native and depleted preparations were resupplied with exogenous Ca²⁺ and Cl^-, we obtained a reduction of atrazine inhibition which was much stronger in the depleted preparations than in the native ones. It is concluded that removal of 16 and 23 kDa polypeptides in general, and disorganization of associated Ca²⁺ and Cl^- in particular, enhances atrazine penetration to its sites of action in the vicinity of the PS II complex. The above could be interpreted if we assume a reduced plastoquinone affinity at the Q_B (secondary plastoquinone electron acceptor) pocket of D1 polypeptide following transmembranous modifications caused by the depletion of these polypeptides.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - Chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - MES 2-(N-morpholino)ethanesulfonic acid - PMSF phenylmethylsul-phonyfluoride - PS II photosystem II - PAGE polyacrilamide gel electrophoresis     

The role of calcium in the pH-dependent control of Photosystem II

pH-dependent inactivation of Photosystem (PS) II and related quenching of chlorophyll-a-fluorescence have been investigated in isolated thylakoids and PS II-particles and related to calcium release at the donor side of PS II. The capacity of oxygen evolution (measured under light saturation) decreases when the pH is high and the pH in the thylakoid lumen decreases below 5.5. Oxygen evolution recovers upon uncoupling. The pH-response of inactivation can be described by a 1 H⁺-transition with an apparent pK-value of about 4.7. The yield of variable fluorescence decreases in parallel to the inactivation of oxygen evolution. pH-dependent quenching requires light and can be inhibited by DCMU. In PS II-particles, inactivation is accompanied by a reversible release of Ca²⁺-ions (one Ca²⁺ released per 200 Chl). In isolated thylakoids, where a pH was created by ATP-hydrolysis, both inactivation of oxygen evolution (and related fluorescence quenching) by internal acidification and the recovery of that inactivation can be suppressed by calcium-channel blockers. In the presence of the Ca²⁺-ionophore A23187, recovery of Chl-fluorescence (after relaxation of the pH) is stimulated by external Ca²⁺ and retarded by EGTA. As shown previously (Krieger and Weis 1993), inactivation of oxygen evolution at low pH is accompanied by an upward shift of the midpoint redox-potential, E_m, of Q_A. Here, we show that in isolated PS II particles the pH-dependent redox-shift (about 160 mV, as measured from redox titration of Chl-fluorescence) is suppressed by Ca²⁺-channel blockers and DCMU. When a redox potential of –80 to –120mV was established in a suspension of isolated thylakoids, the primary quinone acceptor, Q_A, was largely reduced in presence of a pH (created by ATP-hydrolysis) but oxidized in presence of an uncoupler. Ca²⁺-binding at the lumen side seems to control redox processes at the lumen- and stroma-side of PS II. We discuss Ca²⁺-release to be involved in the physiological process of high energy quenching.     

EPR studies of copper(II) and cobalt(II) complexes of adriamycin

Cu2+ and Co2+ complexes of adriamycin (ADM) in aqueous solutions have been examined using EPR spectroscopy. An appreciable amount of Cu2+ and Co2+ complexes formed in the solutions were found to be in the EPR silent associated form, where the metal ions are antiferromagnetically coupled. The associated form of the Cu2+ complex may be neither a simple dimer nor coordination polymer but aggregates of a stacked type. Formation of a complex having Cu2+-ADM stoichiometry of 1:2 was observed for the solutions containing excess of ADM as an EPR observable species. The complex having Cu2+-ADM stoichiometry of 1:1 was not observed directly by EPR, but the presence of the complex is undeniable, especially at low pH range so far as large excessive ADM is not present. The Co2+ complex of ADM observed by EPR is in the high-spin (S = 3/2) state and may have a coordination structure of tetragonal symmetry. The EPR spectra of these complexes apparently show that the Cu2+ and Co2+ ions are bound at the carbonyl and phenolate oxygen in the 1,4-dihydroxyanthraquinone moiety and the amino nitrogen in the sugar part does not seem to participate in the coordination to the metal ions.     

Photoinactivation of the reactivation capacity of photosystem II in pea subchloroplast particles after a complete removal of manganese

After a complete removal of Mn from pea subchloroplast photosystem-II (PS II) preparations the electron phototransfer and oxygen evolution are restored upon addition of Mn²⁺ and Ca²⁺. Pre-illumination of the sample in the absence of Mn²⁺ leads to photoinhibition (PI) — irreversible loss of the capability of PS II to be reactivated by Mn²⁺. The effect of PI is considerably decreased in the presence of Mn²⁺ (4 Mn atoms per reaction center of PS II) and it is increased in the presence of ferricyanide or p-benzoquinone revealing the oxidative nature of the photoeffect. PI results in suppression of oxygen evolution, variable fluorescence, photoreduction of 2,6-dichlorophenol indophenol from either water or diphenylcarbazide. However, photooxidation of chlorophyll P₆₈₀, the primary electron donor of PS II as well as dark and photoinduced EPR signal II (ascribed to secondary electron donors D ₁ and Z) are preserved. PI is accompanied by photooxidation of 2–3 carotenoid molecules per PS II reaction center (RC) that is accelerated in the presence of ferricyanide and is inhibited upon addition of Mn²⁺ or diuron. The conclusion is made that PI in the absence of Mn leads to irreversible oxidative inactivation of electron transfer from water to RC of PS II which remains photochemically active. A loss of functional interaction of RC with the electron transport chain as a common feature for different types of PS II photoinhibition is discussed.Abbreviations A photoinduced absorbance changes - DPC diphenylcarbazide - DPIP 2,6-dichlorophenol indophenol - F _o constant fluorescence of chlorophyll - F photoinduced changes of Chl fluorescence yield - Mn manganese - P₆₈₀ the primary electron donor in PS II - PI photoinhibition - PS II photosystem II - Q the primary (quinone) electron acceptor in PS II - RC reaction center     

Effects of copper and zinc ions on photosystem II studied by EPR spectroscopy.

The effect of Zn(2+) or Cu(2+) ions on Mn-depleted photosystem II (PS II) has been investigated using EPR spectroscopy. In Zn(2+)-treated and Cu(2+)-treated PS II, chemical reduction with sodium dithionite gives rise to a signal attributed to the plastosemiquinone, Q(A)(*)(-), the usual interaction with the non-heme iron being lost. The signal was identified by Q-band EPR spectroscopy which partially resolves the typical g-anisotropy of the semiquinone anion radical. Illumination at 200 K of the unreduced samples gives rise to a single organic free radical in Cu(2+)-treated PS II, and this is assigned to a monomeric chlorophyll cation radical, Chl a(*)(+), based on its (1)H-ENDOR spectrum. The Zn(2+)-treated PS II under the same conditions gives rise to two radical signals present in equal amounts and attributed to the Chl a(*)(+) and the Q(A)(*)(-) formed by light-induced charge separation. When the Cu(2+)-treated PS II is reduced by sodium ascorbate, at >/=77 K electron donation eliminates the donor-side radical leaving the Q(A)(*)(-) EPR signal. The data are explained as follows: (1) Cu(2+) and Zn(2+) have similar effects on PS II (although higher concentrations of Zn(2+) are required) causing the displacement of the non-heme Fe(2+). (2) In both cases chlorophyll is the electron donor at 200 K. It is proposed that the lack of a light-induced Q(A)(*)(-) signal in the unreduced Cu(2+)-treated sample is due to Cu(2+) acting as an electron acceptor from Q(A)(*)(-) at low temperature, forming the Cu(+) state and leaving the electron donor radical Chl a(*)(+) detectable by EPR. (3) The Cu(2+) in PS II is chemically reducible by ascorbate prior to illumination, and the metal can therefore no longer act as an electron acceptor; thus Q(A)(*)(-) is generated by illumination in such samples. (4) With dithionite, both the Cu(2+) and the quinone are reduced resulting in the presence of Q(A)(*)(-) in the dark. The suggested high redox potential of Cu(2+) when in the Fe(2+) site in PS II is in contrast to the situation in the bacterial reaction center where it has been shown in earlier work that the Cu(2+) is unreduced by dithionite. It cannot be ruled out however that Q(A)-Cu(2+) is formed and a magnetic interaction is responsible for the lack of the Q(A)(-) signal when no exogenous reductant is present. With this alternative possibility, the effects of reductants would be explained as the loss of Cu(2+) (due to formation of Cu(+)) leading to loss of the Cu(2+) from the Fe(2+) site due to the binding equilibrium. The quite different binding and redox behavior of the metal in the iron site in PS II compared to that of the bacterial reaction center is presumably a further reflection of the differences in the coordination of the iron in the two systems.     

Photosystem II electron transfer cycle and chlororespiration in planktonic diatoms

The dominance of diatoms in turbulent waters suggests special adaptations to the wide fluctuations in light intensity that phytoplankton must cope with in such an environment. Our recent demonstration of the unusually effective photoprotection by the xanthophyll cycle in diatoms [Lavaud et al. (2002) Plant Physiol 129 (3) (in press)] also revealed that failure of this protection led to inactivation of oxygen evolution, but not to the expected photoinhibition. Photo-oxidative damage might be prevented by an electron transfer cycle around Photosystem II (PS II). The induction of such a cycle at high light intensity was verified by measurements of the flash number dependence of oxygen production in a series of single-turnover flashes. After a few minutes of saturating illumination, the oxygen flash yields are temporarily decreased. The deficit in oxygen production amounts to at most 3 electrons per PS II, but continues to reappear with a half time of 2 min in the dark until the total pool of reducing equivalents accumulated during the illumination has been consumed by (chloro)respiration. This is attributed to an electron transfer pathway from the plastoquinone pool or the acceptor side of PS II to the donor side of PS II that is insignificant at limiting light intensity but is accelerated to milliseconds at excess light intensity. Partial filling of the 3-equivalents capacity of the cyclic electron transfer path in PS II may prevent both acceptor-side photoinhibition in oxygen-evolving PS II and donor-side photoinhibition when the oxygen-evolving complex is temporarily inactivated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Binding of zinc(II) and copper(II) to the full-length Alzheimer's amyloid-beta peptide

There is evidence that binding of metal ions like Zn2+ and Cu2+ to amyloid beta-peptides (Abeta) may contribute to the pathogenesis of Alzheimer's disease. Cu2+ and Zn2+ form complexes with Abeta peptides in vitro; however, the published metal-binding affinities of Abeta vary in an enormously large range. We studied the interactions of Cu2+ and Zn2+ with monomeric Abeta(40) under different conditions using intrinsic Abeta fluorescence and metal-selective fluorescent dyes. We showed that Cu(2+) forms a stable and soluble 1 : 1 complex with Abeta(40), however, buffer compounds act as competitive copper-binding ligands and affect the apparent K(D). Buffer-independent conditional K(D) for Cu(II)-Abeta(40) complex at pH 7.4 is equal to 0.035 micromol/L. Interaction of Abeta(40) with Zn2+ is more complicated as partial aggregation of the peptide occurs during zinc titration experiment and in the same time period (within 30 min) the initial Zn-Abeta(40) complex (K(D) = 60 micromol/L) undergoes a transition to a more tight complex with K(D) approximately 2 micromol/L. Competition of Abeta(40) with ion-selective fluorescent dyes Phen Green and Zincon showed that the K(D) values determined from intrinsic fluorescence of Abeta correspond to the binding of the first Cu2+ and Zn2+ ions to the peptide with the highest affinity. Interaction of both Zn2+ and Cu2+ ions with Abeta peptides may occur in brain areas affected by Alzheimer's disease and Zn2+-induced transition in the peptide structure might contribute to amyloid plaque formation.     

Copper(II) facilitates bleomycin-mediated unwinding of plasmid DNA

The unwinding of plasmid DNA by bleomycin A2 (BLM A2) was investigated by use of two-dimensional gel electrophoresis. It was found that Cu2+ ions greatly facilitated the unwinding of topoisomers of plasmid DNA by BLM A2 at concentrations where cupric ions alone had no effect on DNA supercoiling. The concentration of BLM A2 required for observable unwinding was reduced at least 100-fold in the presence of equimolar Cu2+. A plot of [Cu2+] vs extent of DNA unwinding in the presence of 10(-4) M BLM A2 gave a curve consistent with the action of cupric ions on BLM in an allosteric fashion, possibly rearranging the drug into a conformation that facilitates DNA unwinding. The participation of the metal center in enhancing DNA unwinding via direct ionic interaction with one or more negatively charged groups on the DNA duplex also seems possible. Further analysis of the structural factors required for BLM-mediated DNA unwinding was carried out with Cu2+ + BLM demethyl A2, the latter of which differs from BLM A2 only in that it lacks a methyl group, and associated positive charge, at the C-terminus. Cu(II).BLM demethyl A2 was found to be much less effective than Cu(II).BLM A2 as a DNA unwinding agent, emphasizing the strong dependence of this process on the presence of positively charged groups within the BLM molecule. These findings constitute the first direct evidence that the metal center of BLM can participate in DNA interaction, as well as in the previously recognized role of oxygen binding and activation.     

Effect of copper ions on the light-induced proton transfer in spinach chloroplasts

It was shown that the light-dependent proton uptake by a suspension of isolated chloroplasts was completely inhibited in the presence of 30-50 microM Cu2+ ions at the 0.1-0.3 Cu2+/Chl ratio. At the same time, the rate of photosynthetic oxygen evolution in the presence of 30-200 microM CuSO4 was reduced by no more than 20-30% of control and up to 50% of the control DeltapH value was retained. The results allow us to suppose that, in the presence of copper ions: 1) electron transport in PS2 is inhibited at the level of the secondary quinone acceptor Q(B) whose photoreduction is accompanied by proton uptake from external medium; and 2) an alternative pathway of electron transfer to terminal acceptor is activated, which provides the photooxidation of water and the formation of transmembrane proton gradient.